Electrophoresis is a process for separation of charged molecules and exploits the different mobility of molecules in an electric field. The mobility of molecules in an electric field depends on several factors such as the electrophoretic medium, the electric field strength and the characteristics of molecules (ions) themselves, such as size, shape and charge density. Electrophoresis is primarily used for the separation of biological macromolecules, such as proteins, nucleic acids and their derivatives. The process is usually carried out by forcing the molecules to migrate through a gel-supporting medium, the latter serving as a molecular sieve that enhances separation. The gel may be composed of natural or synthetic polymers. Agarose is the most widely used natural material and polyacrylamide gels represent the most common synthetic matrix. The gel may be immersed in a buffer, which serves as a conductive medium between electrodes and the gel. This format is known as submerged gel electrophoresis and it is the simplest to operate. Submerged gel electrophoresis is widely used for the analysis of nucleic acids.
Gels for electrophoresis can be prepared by free radical polymerization, by thermally induced gelation, and by a cross-linking reaction taking place simultaneously with gelation. Each of the three processes is currently being used for the production of precast gels for electrophoresis. Precast electrophoresis gels are manufactured by outside vendors, and are then shipped to the laboratory where the electrophoresis will be performed. Precast gels must have the required properties for electrophoresis and they must be able to maintain these properties throughout shipping and storage. The shelf life of many precast gels is limited by the potential for hydrolysis of acrylamide and/or buffer constitution during storage at the high pH of the gel buffer. Neutral buffer systems reduce degradation of polyacrylamide gels by hydrolysis, thereby increasing the useful shelf life of precast gels, as well as increasing the stability of the gels during electrophoresis. However, neutral gel systems suffer the disadvantage of providing lower separation and/or resolution.
Cross-linked polyacrylamide, produced by polymerizing acrylamide containing a few percent of N,N′-methylenebisacrylamide, is extensively employed as the matrix for gel electrophoresis. This is due primarily to the properties of the polymer, namely: excellent mechanical strength, adherence to glass surfaces, and a pore size which is controllable over a wide range. Polyacrylamide gels provide for high resolution of small sized sample components, e.g. they are capable of providing high resolution of DNA ranging in size from 6 to 1000 bp in length. Other advantages of cross-linked polyacrylamide gels are that they are optically transparent, providing for easy identification of separated sample components, they do not bind charged analytes, and they do not engender electroosmotic flow.
There are, however, certain properties of cross-linked polyacrylamide, which detract from its application as an electrophoretic medium. A major problem is that the cross-linked polyacrylamide gel must be prepared in situ, i.e., directly in an electrophoresis chamber utilizing free radical polymerization. Free radical reactions depend on a variety of parameters such as concentration of initiator, monomer purity, temperature, time, and absence of oxygen and other inhibitors. Managing these factors can require an inordinate amount of care and attention in order to achieve reproducible results. Another disadvantage associated with cross-linked polyacrylamide is the possible health hazard from handling the precursor monomers, acrylamide having been found to be a neurotoxin. A further disadvantage is that polyacrylamide gels are subject to degradation by hydrolysis and have a limited shelf life. In this regard, polyacrylamide gels are usually poured and run at basic pH. Hydrolysis of polyacrylamide at basic pH proceeds even at low temperature and the carboxylic groups formed are incorporated into the polymer, thus generating unwanted electro-endosmosis during electrophoresis.
Over the past two decades, a great deal of effort has been expended in the investigation and development of electrophoretic gel systems, which are free of the problems associated with polyacrylamide. In this regard, the use of agarose, a polysaccharide, showed considerable promise in the field of electrophoresis. Agarose gels, which comprise a natural, substantially linear, alternating copolymer of beta-D-galactose and 3,6-anhydro-alpha-L-galactose in an electrophoresis buffer, have many advantages in electrophoresis. They are thermoreversible, i.e. they undergo a transition from a first flowable state to second gel state in response to a change in temperature, thereby enabling separated components to be recovered from the melted gel. Agarose gels are also easy to prepare since they do not require free radical polymerization for gel formation. Furthermore, agarose is non-toxic, has a low susceptibility to electro-endoosmosis and has a high mechanical gel strength, providing for ease of manipulation. Gels prepared with native agarose exhibit a characteristic coarse pore structure, a feature that renders them the preferred medium for the electrophoretic separation of large macromolecules. Agarose gels comprising about 0.6 to 1 percent polymer are suitable primarily for the separation of proteins having molecular weights in excess of about 500,000 (500 kD) and DNA molecules in the size range from a few hundred to a few tens of thousand base pairs.
Despite these advantages, there are a number of disadvantages associated with the use of agarose gels in electrophoretic separation media. Smaller DNA molecules require higher agarose concentrations for good resolution, as generally known in the prior art. However, more concentrated agarose gels are difficult to prepare due to the resulting high viscosity of the agarose solutions. Furthermore, visualization of separated bands is difficult due to gel opacity.
It is known from the prior art that a gel of a particular composition and of a certain total concentration gives optimal resolution of macromolecules only in a limited size range. It is general knowledge in the art that outside a certain size range the resolution will be poor. Smaller molecules will give broad bands, whereas larger ones will not migrate sufficiently to be separated in a meaningful running time. Thus, the resolving power in the lower size range is limited by separation efficiency, and in the upper size range by separation selectivity. The resolving power of a gel can be improved by enhancing the efficiency or selectivity, or both.
The large pore limitation of agarose gels can be diminished and their sieving action improved by forming the gels from certain agarose derivatives having a finer pore structure than the parent agarose. U.S. Pat. No. 3,956,273 to Guisely and U.S. Pat. No. 4,319,975 to Cook disclose many derivatized agarose polymers. Although the majority of reagents used for derivatization were monofunctional, a bifunctional reagent could also be used. However, Guisely teaches that the ratio of the bifunctional reagent to agarose, as well as derivatization conditions, must be such that the polymer chains are not cross-linked, since otherwise the resulting product could not be re-dissolved, as required for a subsequent preparation of the electrophoresis gel. One preferred class of such modified agarose is hydroxyalkylated agarose produced by replacing hydroxyl groups 1 to 4 in the agarobiose units of the agarose polymer chain with hydroxyalkyl moieties.
A preferred member of this class, hydroxyethylated agarose, is obtained by reacting agarose with 2-chloroethanol in the presence of alkali. Gels formed from hydroxyethylated agarose are capable of resolving proteins from about 50 kD to about 600 kD. Moreover, such gels have lower melting points than native agarose gels, an advantage when recovering sensitive biological substances from the re-melted gels. Derivatization of hydroxyl groups of agarose, as disclosed in U.S. Pat. No. 3,956,273, reduces viscosity of agarose solutions, as well as gel opacity. Such hydroxyethylated agarose derivatives are commercially available products known under the trade name SeaPlaque (RTM) and NuSieve (RTM) (FMC Corporation). NuSieve (RTM) agarose that contains partially depolymerized hydroxyalkylagarose is typically used at polymer concentrations from about 2 to 8%, and improved resolution of small DNA using this agarose has been reported [Dumais and Nochumson, Bio Techniques, 5 (1987) 62]. However, separated DNA bands were bent (WO 92/15868 to Kozulic). The bending could be reduced by adjustment of ionic composition of the gel. However, it would be preferable to have a gel that does not require such an adjustment.
An aqueous electrophoretic resolving gel composition comprising two polysaccharide hydrogels, at least one of which has been derivatized, and independently, at least one of which has been partially de-polymerized sufficiently to reduce the casting-effective viscosity of the gel composition, has been proposed (U.S. Pat. No. 5,143,646 to Nochumson). In the preferred embodiment of the invention, component 1 of the resolving gel was partially de-polymerized hydroxyalkylated agarose while component 2 was a 1,2-dihydroxypropyl derivative of agarose (glycerated agarose). Some loss of sieving efficiency was incurred by the de-polymerized resolving gel component but this was compensated by the presence of a non-depolymerized component. The total amount of the two polysaccharides in the resolving gel was 4 to 8 wt. %. The gel system of this invention afforded separation of proteins in the molecular weight range of 10 to 200 kD with good resolution of individual protein bands, moderate viscosity for easy handling and melting of the gels at low temperature for easy sample recovery.
Bands representing separated species are bent in a submerged electrophoresis gel containing a high polymer concentration and the same buffer as the running buffer. The bending effect is related largely to the resistance of polymer chains to migration of buffer ions and is also possibly due to a change of relative migration rates of ions in the gel and buffer. The resistance may be reduced by lowering the polymer concentration of the gel. In an attempt to produce gels with a suitable polymer concentration, cross-linked agarose derivatives have been prepared with the assumption that cross-linking will improve the stability and sieving efficiency of the gel.
Several different cross-linked agarose derivatives are known in the prior art. U.S. Pat. No. 3,507,851 to Ghetie discloses cross-linking of agarose particles with epichlorhydrin. U.S. Pat. No. 3,959,251 and UK Patent No. 1,352,613 both to Porath et al. disclose stabilization of agarose beads by cross-linking with several bifunctional reagents in the presence of a reducing agent. The resulting beads had greater rigidity, giving higher flow rates when packed into columns for chromatography. The cross-linking reaction was carried out after the gel was formed and the resulting products were in the form of particles.
Cross-linked agarose gels in the form of plates are also known. U.S. Pat. No. 3,860,573 to Honkanen discloses agarose gels cross-linked with a bifunctional reagent containing two equal functional groups selected from acyl chloride, sulfonyl chloride and isothiocyanate. A process for treating polysaccharide gels comprising suspending or dissolving the polysaccharide gel in a solution of 2,4,6-trichloro-1,3,5-triazine, is disclosed in U.S. Pat. No. 3,956,272 to Tixier. However, the cross-linking reactions of Honkanen and Tixier introduced charged groups into the gel-forming polymer.
Honkanen and Tixier reported that their cross-linked agarose gels could be used as media for electrophoresis. Regarding properties of these gel matrices, Honkenen teaches that movement of large molecules (proteins) is more rapid in the cross-linked agarose, which would indicate an increase of effective porosity due to the cross-linking reaction. Tixier discloses that the treated gels had essentially the same characteristics as untreated gels with respect to sieving and resolving power. This result of Tixier is in accordance with the report of Porath (J. Chomatogr. 103 (1975) 49-62) who found that cross-linked agarose beads did not change their porosity.
Gels formed by reacting many different polymers/cross-linker combinations under various cross-linking conditions are described in U.S. Pat. No. 5,541,255 to Kozulic. The cross-linkers react with hydroxyl groups of polymers to form ether linkages without introducing a charged group into the gel. It is possible to use natural or synthetic polymers possessing hydroxyl groups but natural polymers are preferred because of their pronounced hydrophilicity. The most common of such polymers is agarose. Compounds having oxirane or halo groups are particularly suitable as possible cross-linkers. Examples of the cross-linkers include epichlorohidrin, butanediol diglycidylether and 1,2-dibromopropanol. The gel formation and cross-linking reactions proceed simultaneously in a water solution of dissolved agarose. Because of the high ratio of the cross-linker to polysaccharide, the gel is always in the form of a continuous water insoluble bed.
The cross-linked agarose gels described by Kozulic were used to support electrophoresis. The separation range of the gels depended on the polymer type, its concentration, cross-linker type, and cross-linker concentration, as well as the cross-linking conditions. For example, a gel containing 1% of cross-linked agarose gave very good separation of DNA fragments in the size range from about 150 to 3000 bp. Improved resolution of small (200-600 bp) DNA molecules was achieved, as compared to results obtained by using a native agarose polymer. Moreover, the gels formed according to the above invention were soft and elastic and they had improved transparency. However, a main drawback of these cross-linked agarose gels is their impurity. In this regard, the formed gels contain some unreacted cross-linker, byproducts of the gelation reaction, and the base which is used as a catalyst. Accordingly, to produce a gel of well-defined ionic composition, it is necessary to firstly wash the cross-linked gel in water, and then incubate the gel in a desirable electrophoresis buffer. A further draw back arising from the cross-linked gels described by Kozulic is that the gels are not reversible, thus preventing separated components from being recovered.
An alternative way of varying the properties of gels suitable for use in electrophoresis, is to add an additive into a formed gel or into a gelling solution. The additive is chosen such that it improves a particular property of the gel to which it is added. In this regard, additives have been used in combination with agarose gels. As mentioned above, agarose gels have inferior optical properties compared to polyacrylamide gels. This drawback can be partially corrected by adding another polysaccharide into the agarose solution prior to its gelation (U.S. Pat. No. 5,230,832 to Perlman). The sieving properties of agarose gels can be improved by combining them with other gel forming materials such as polyacrylamide (Bode, H. J. (1977) Anal. Biochem. 83, 204-210; Horowitz, P. M. et all. (1984) Anal. Biochem. 143, 333-340). In these gels, the agarose provided mechanical stability, whereas the polyacrylamide served as a sieving medium. Another example includes the addition of polyethylene glycol (PEG) into a pre-formed gel, such as a cellulose acetate gel. In this combination the cellulose acetate served as the stabilizing medium whereas the PEG was the sieving medium (Bode, H. J. (1976) FEBS Letters. 65, 56-58). However, such mixtures have compatibility problems, especially when they contain high percentages of agarose. Moreover, such heterogeneous agarose blends have not afforded a consistent improvement of protein separation patterns. This is particularly so in the context of submerged electrophoresis, where additives not incorporated in the gel structure have a tendency to diffuse out of the gel.
Electrophoresis gels of enhanced selectivity can be produced by adding a preformed polymer (additive) to a polymerization solution containing acrylamide or (N-acryloyl-tris(hydroxymethyl)aminomethane (NAT) and a cross-linker (U.S. Pat. No. 5,840,877 to Kozulic). Agarose, hydroxyethylcellulose and other polysaccharides can be used as preformed polymers. In this connection, it has been noticed that the relationship between the polymers formed by free-radical polymerization in the presence of an additive is complex, since in some instances the newly formed polymer and the preformed additive will become covalently linked by a chain transfer reaction during free-radical polymerization. Moreover, a polymeric additive may be intertwined with the gelled polymers so strongly that for all practical applications it may be considered an integral part of the gel matrix. Alternatively, the additive may be only loosely associated with gel polymers, or it may just remain in the gel interstices, allowing easy diffusion out of the gel. It was found that at a specific ratio of additive to monomer and cross-linker, an enhanced selectivity of the gel was achieved, i.e., the electrophoretic migration of larger molecules was retarded relative to the electrophoretic migration of the smaller molecules. This reduction might be related to the high friction between gel polymers.
Other polysaccharides have been used as additives in polymerization solutions, with the intention of enhancing selectivity of electrophoretic separations. These include dextran, the polysaccharide from locust bean, carubin-type galactomannan (U.S. Pat. No. 5,840,877 to Kozulic; WO 0077059 to Lazar). Such polysaccharides have given rise to a selectivity enhancement, enabling fine resolution on a much shorter gel length than previously possible.
Synthetic polymers have also been added to polymerization solutions of acrylamide or NAT (U.S. Pat. No. 5,840,877 to Kozulic). In this regard, PEG, polyvinyl alcohol and polyvinylpyrrolidone were not able to give gels of enhanced selectivity. However, there was a strong retardation of larger DNA molecules in gels containing linear polyacrylamide.
Graft polymerization involves reacting a monomer with a polymer substrate. Graft polymerization depends on the creation of active sites on the substrate. In free radical-initiated chemical grafting, active sites are usually created as a result of a hydrogen abstraction reaction. The polymerization reaction is thus initiated directly by a polymer chain radical, and when unsaturated monomer is present, polymerization occurs, resulting in the newly formed polymer being covalently bonded or grafted onto the existing polymer molecule.
A number of chemical activators are known. The production of starch graft copolymers or cellulose graft copolymers utilizing polymerization initiators such as hydrogen peroxide, organic peroxides and hydroperoxides have been reported. Yields of grafted chains may be improved by the use of an activator for these initiators such as mild reducing agents, e.g., ferrous ammonium sulfate, sodium sulphoxylate and the like. For the most part, these initiators are nonspecific and induce homopolymerization of single monomers and copolymerization of monomer mixtures, as well as the desired graft polymerization of monomer and monomer mixtures to the substrate. This produces products which tend to separate on storage.
Such problems can be minimized or avoided by the use of a cerium (4+) ammonium salt as an initiator. The cerium ion (4+) interacts with various protonated organic groups, including hydroxyl, carboxyl, amine, etc., to remove an electron to the metal ion, and leave an initiating radical behind on the organic group. In such a system, the metal ion acts as an oxidizer. Thus, the cerium (4+) salt forms a redox pair with an organic reducing agent. For example, U.S. Pat. No. 2,922,768 to Mino et al. teaches the polymerization of vinyl monomers in the presence of cerium salt with organic reducing agents, such as alcohols, aldehydes, thiols, glycols and amines. If a polymeric reducing agent such as a polysaccharide or a poly(vinyl alcohol) is employed, and the oxidation is conducted in the presence of a vinyl or olefin monomer, graft polymerization will occur on the substrate. Although some homopolymerization has been reported using cerium (4+) by Fanta et al. (J. Appl. Polymer Sci., Vol. 10, pp. 919-937, 1966), the most important pathway for cerium (4+) initiation of free radicals as outlined by Fanta (Block and Graft Copolymers, Vol. 1, pp. 1-45, Ed. R. J. Ceresa, John Wiley & Sons, London & New York, 1973) would be expected to give graft copolymers to the exclusion of any homo- or copolymers. Extensive use has been made of this system to graft vinyl monomers to starch, cellulose, dextran, poly(vinyl alcohol) and other polymers possessing hydroxyl groups. The graft copolymerization of vinyl monomers onto cellulose in the presence of cerium salts proceeds readily when the monomer is a polar electron acceptor monomer such as acrylonitrile or acrylates.
The chemistry of polymer-grafting onto polymers containing hydroxyl groups in the presence of cerium (4+) salts has been discussed in the literature (G. Odian, J. Kho. Macromol. Sci. Chem. A4(2) (1970) 317-330). The chain growth radical polymerization is initiated by the abstraction of a hydrogen atom from such a polymer, which is then oxidized to a hydrogen ion via a reversible cerium ion redox reaction (G. Mino and S. Kaizerman. J. Polym. Sci. 31 (1958) 242-243). The activated polymers carry free radicals that rapidly react with vinyl groups of monomers present in solution that initiate polymerization, exclusively on the polymer backbone.
A method of graft polymerization for producing phase supports for partition chromatography is described in U.S. Pat. No. 4,756,834 to Muller et al. The phase support comprises base support particles consisting of an inorganic and/or organic material and an extensive list of such materials is disclosed. A surface layer of polymeric material, preferably polyacrylamide, is attached to the base support particles by graft polymerization. Cerium (IV) ions can be used as the polymerization catalyst. This patent is not concerned with electrophoretic gels. Graft polymerization is also used in U.S. Pat. Nos. 5,674,946 and 6,291,216 both to Muller to produce activated support materials based on hydroxyl-containing base supports for use in chromatographic separating materials and for immobilization of enzymes. The surfaces of the hydroxyl-containing base supports are covalently bonded to polymers by graft polymerization. The reaction is carried out in the presence of Cerium (IV) ions. Suitable base support listed are agarose-based polysaccharides, cellulose, cellulose derivatives and polymers based on dextran. Acryloylated chlorohydrin-containing alkyl-amines are polymerized onto the base supports in U.S. Pat. No. 5,674,946 and vinyl monomers are polymerized onto the base supports in U.S. Pat No. 6,291,216. These patents are not concerned with the production of a gel for electophoresis.
It has been found recently that monomers such as poly(ethylene glycol)acrylate, poly(propylene glycol)acrylate, glycerol acrylate, hydroxyalkyl acrylate, poly(ethylene glycol)methacrylate, poly(propylene glycol)methacrylate, glycerol methacrylate and /hydroxyalkyl methacrylates may be used to form polymers that are well-suited for use as electrophoresis support media (U.S. Pat. No. 5,290,411 to Zewert and Harrington). In this regard, alkylene glycol esters of methyacrylic or acrylic acid have been polymerized and cross-linked to different degrees to provide electrophoresis support media ranging from viscous liquids to gels. If desired, these monomers may be copolymerized with acrylamide. In general, the resolution with the resulting gels was of the same order as that obtainable with polyacrylamide. Some gels gave sharper banding than polyacrylamide. Moreover, the use of such monomers has the significant advantage in that they are considerably less toxic than acrylamide. However, these gels have a number of disadvantages (when used in an aqueous environment) compared to acrylamide gels. For example, the gels are not as mechanically robust as acrylamide gels (when using an equivalent amount of cross-linker). Furthermore, the rate of protein migration through the gels is significantly slower than that for acrylamide gels.
The present invention addresses the disadvantages associated with the above prior art.